MethodsX 1 (2014) 264–268

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Analysis of coffee bean extracts by use of ultra-performance

liquid chromatography coupled to quadrupole time-of-flightmass spectrometry

Daniel James O’Driscoll *

Alimentary Pharmabiotic Centre, Biosciences Institute, University College Cork, Cork, Ireland

G R A P H I C A L A B S T R A C T

A B S T R A C T

The number of flavour chemicals identified in coffee has reached over 1000 [1,2]. Coffee is one of the world’s most popular beverages [3],

highly studied for its health-related properties [4–6]. Studies on coffee associated with human health have focused on the negative

aspects, such as the toxicity of caffeine [7,8]. Complex chemistry happens during coffee roasting and according to the literature, a number

of compounds have been detected and quantified in coffee beans samples by UPLC–Q-TOF/MS [9–12]. The following method offers a

simple approach for the qualitative and quantitative analysis of coffee bean extracts using a Waters Acquity G2 UPLC–Q-TOF/MS

instrument adapted from the method by Kenny et al., [12]. The following modifications were made:

� The method by Kenny et al. was developed on a triple quadrupole mass spectrometer, the below method was developed on a Q-TOF MS.

� A combination of utilising both base peak index and mass extraction at 0.05 Da allows for a sensitive, quantitative technique amidst

poor background noise and poor separation with high mass accuracy (<5 ppm).

� By use of MSE centroid experiment, greater mass spectral information for metabolite profiling could be obtained.

� 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/

licenses/by/3.0/).

A R T I C L E I N F O

Method name: Analysis of coffee bean extracts by use of ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Keywords: UPLC, Q-TOF/MS, Caffeine, Mass spectrometry, Phenolic analysis, WatersArticle history: Received 5 August 2014; Accepted 24 October 2014; Available online 3 November 2014

* Tel.: +353 021 4902931; fax: +353 21 427 0244.

E-mail address: daniel.odriscoll@ucc.ie

http://dx.doi.org/10.1016/j.mex.2014.10.006

2215-0161/� 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/

3.0/).

D.J. O’Driscoll / MethodsX 1 (2014) 264–268 265

Method details

Liquid chromatography mass spectrometry analysis

The analysis was performed using a Waters Acquity G2 Q-TOF LC–MS instrument. This system is composed of aWaters Acquity UPLC system coupled to a quadrupole time-of flight mass spectrometer. The samples were eluted usinga Titan C18 HPLC analytical column (100 mm� 2.1 mm, 1.9 mm) and preceded by a Titan C18 guard cartridge(5 mm� 2.1 mm, 1.9 mm) with the column set to 35 8C. All samples were kept refrigerated to 4 8C in the UPLCautosampler and a 10 mL injection volume was used with a total flow rate of 0.3 mL/min over a total run time of 12 min.All solvents used were LC–MS grade and ultra-pure 18.2 MV water was used for each step. Mobile phase A consisted ofwater + 0.1% formic acid while mobile phase B was acetonitrile + 0.1% formic acid. The following tables contain thegradient details for list of compounds analysed (Tables 1 and 2):

Mass spectrometry detection was conducted through electrospray ionisation using an msE centroid experiment inboth positive and negative mode and screened in the m/z scan range of 50–2000 Da (Table 3) with the analyser set toresolution mode at FWHM. Scanning conditions were set to 1 scan every 0.7 s. Collision energy was set for two functions,function one at low energy with no collision energy applied and function two at high energy using a collision energyramp from 20 to 75 eV. In negative mode the following MS tune file settings were used: Capillary voltage 3.00 kV,sampling cone 40 V, extraction cone 4.0 V, source temperature 120 8C, desolvation temperature 450 8C, desolvation gasflow 800 L/h, cone gas flow 50 L/h. In positive mode, the following MS tune file settings were used: capillary voltage3.00 kV, sampling cone 30 V, extraction cone 2.0 V, source temperature 120 8C, desolvation temperature 450 8C,desolvation gas flow 800 L/h, cone gas flow 50 L/h. The accurate mass of the instrument was initially calibrated throughdirect infusion of a sodium iodide calibrant solution prior to sample analysis. In addition, leucine enkephalin (Leuenk)lockmass solution (2 ng/uL) was infused at 5 mL/min in parallel to the mobile phase flow, scanned and automaticallycorrected to verify exact mass which ensured high mass accuracy (<5 ppm) throughout the scan range over the courseof the submitted sequence. Masslynx v4.1 software was used to control the instrument and also analyse the data.

Sample extraction

Coffee beans (green and roasted) were frozen with liquid nitrogen and ground with a mill. Ground coffee samples(2 g) were extracted with LC grade water at 92 8C (25 mL) then stirred for 6 min at 70–80 8C and placed on iceimmediately after in order to cool down rapidly. The samples were centrifuge at 21,481� g for 2 min. Aftercentrifugation the extracts were filtered through a 0.2 mm PVDF membrane. Extracts were poured into 1.5 mL vialsand sealed. All other remaining samples and extracts were kept in the freezer at �20 8C.

Table 1

Gradient used for separation and identification of standards reconstituted in water include quinic acid, ferulic acid,

pyrogallol, and trigonelline hydrochloride.

Time (min) Flow rate %A %B

Initial 0.3 98.0 2.0

1 0.3 98.0 2.0

2 0.3 90.0 10.0

3 0.3 80.0 20.0

6 0.3 80.0 20.0

7.5 0.3 65.0 35.0

8.5 0.3 10.0 90.0

9.5 0.3 10.0 90.0

12 0.3 98.0 2.0

Table 2

Gradient used for separation and identification of standards reconstituted in MeOH include caffeine, 5-caffeoylquinic

acid, vitamin B3, caffeic acid, catechol, and 1,2,4-benzentriol.

Time (min) Flow rate %A %B

Initial 0.3 98.0 2.0

1 0.3 98.0 2.0

2 0.3 90.0 10.0

3 0.3 90.0 10.0

6 0.3 90.0 10.0

7.5 0.3 50.0 50.0

8.5 0.3 10.0 90.0

9.5 0.3 10.0 90.0

12 0.3 98.0 2.0

Table 3

List of compounds (standards) qualitatively and quantitatively analysed in both positive and negative mode ionisation.

Molecular formula Monoisotopic mass (Da) Compounds Solvent solubility Polarity (+/�) % Error ppm

C8H10N4O2 194.080383 Caffeine MeOH + 3.1

C7H7NO2 137.047684 Trigonelline hydrochloride Water + 5.0

C16H18O9 354.095093 5-Caffeoylquinic acid MeOH � �2.3

C7H12O6 192.063385 Quinic acid Water � �1.0

C6H5NO2 123.032028 Vitamin B3 MeOH + 4.2

C10H10O4 194.057907 Ferulic acid Water � �1.0

C9H8O4 180.042252 Caffeic acid MeOH � �1.7

C6H6O2 110.036781 Catechol MeOH � 0.9

C6H6O3 126.031693 Pyrogallol Water � �1.6

C6H6O3 126.031693 1,2,4-Benzenetriol MeOH � 4.0

Table 4

R2 values for calibration curves (n=8) generated from Waters Acquity G2 UPLC–Q-TOF/MS instrument.

Compounds Concentration range (mg/mL) LOD (mg/mL) LOQ (mg/mL) R2 Sample Green Bean (mg/mL)

Caffeine 0.00036–0.00359 0.0000119 0.0000396 0.9967 0.00040

Trigonelline hydrochloride 0.00047–0.00367 0.0000364 0.0001215 0.9954 0.00202

5-Caffeoylquinic acid 0.00039–0.0028 0.0000185 0.0000637 0.9961 nd

Quinic acid 0.00026–0.00203 0.000020 0.000080 0.9984 0.00143

Vitamin B3 0.00042–0.00328 0.0000185 0.0000617 0.9981 0.0009

Ferulic acid 0.00038–0.00297 0.000020 0.000080 0.9943 0.00100

Caffeic acid 0.00043–0.00336 0.000020 0.000080 0.9974 0.00273

Catechol 0.00015–0.00117 0.000020 0.000080 0.9982 0.00066

Pyrogallol 0.00021–0.00164 0.000020 0.000080 0.9976 0.0003

1,2,4-Benzenetriol 0.0001–0.00781 0.000020 0.000080 0.9966 0.00134

D.J. O’Driscoll / MethodsX 1 (2014) 264–268266

Stock solution preparation

Two stock solutions were prepared, these included methanol and water depending on the solubility of the compound. Allstandards were prepared between 1 and 8 mg to a final volume of 10 mL. The methanol stock solution consisted of caffeine,5-caffeoylquinic acid, vitamin B3, caffeic acid, catechol and 1,2,4-benzenetriol. While the water stock solution consisted oftrigonelline hydrochloride, quinic acid, ferulic acid and pyrogallol.

Quantification

Quantification was performed by generation of suitably linear curves for each of the analysed standards (Table 4). Allstandard curves were created in Microsoft Excel, 2010. For the purpose of this method it was deemed necessary to determine

[(Fig._1)TD$FIG]

Fig. 1. Full scan MS (negative mode) chromatogram conducted in the mass to charge (m/z) range between 50 and 2000 Da displaying elemental composition

and error ppm reconstituted in MeOH.

[(Fig._2)TD$FIG]

Fig. 2. Full scan MS (positive mode) chromatogram conducted in the mass to charge (m/z) range between 50 and 2000 Da displaying elemental composition

and error ppm reconstituted in MeOH.

D.J. O’Driscoll / MethodsX 1 (2014) 264–268 267

only limit of detection (LOD), limit of quantitation (LOQ) and finally linearity over the range to obtain suitable R2 values. Inorder to establish LOD and LOQ values, each analyte was determined as concentrations equivalents to three times and10 times the signal-to-noise ratio of the compounds of interest in the lowest concentration of the calibration curve preparedusing the green bean extract. The signal-to-noise ratios were calculated using Waters MassLynx software version 4.1

Base peak index (BPI) full scan MS chromatographic profiles

Discussion of figures

Figs. 1 and 2 represent total ion count chromatograms generated from the analysis of methanol in both positive andnegative mode mass spectrometry while Figs. 3 and 4 were analysed in water. From the peaks obtained it can be seen thatsome compounds ionise in both positive and negative polarity, labelled peaks represent best ionisation. In Fig. 1, peak atretention time 1.40 represents vitamin B3, in Fig. 2 the peaks at retention time 4.49 and 7.71 represent both 5-caffeoylquinicacid and caffeic acid, respectively. For confidentiality reasons, peak at retention time 4.30 in Fig. 3, peak at retention time1.56 in Figs. 1 and 2 and finally peaks at retention times 4.27 and 6.46 cannot be disclosed.

[(Fig._3)TD$FIG]

Fig. 3. Full scan MS (negative mode) chromatogram conducted in the mass to charge (m/z) range between 50 and 2000 Da displaying elemental composition

and error ppm reconstituted in water.

[(Fig._4)TD$FIG]

Fig. 4. Full scan MS (positive mode) chromatogram conducted in the mass to charge (m/z) range between 50 and 2000 Da displaying elemental composition

and error ppm reconstituted in water.

D.J. O’Driscoll / MethodsX 1 (2014) 264–268268

Acknowledgements

The publication has emanated from research supported in part by a research grant from Science Founfation Ireland(SFI) under Grant Number SFI/12/RC/2273. Funding under the higher education authorities (HEA’s) programme forresearch in third-level institutions (PRTLI) and co-funded under the European Regional Development Fund. The authorwould also like to acknowledge both Camille Delebecque and Sophie Deterre of the Afineur coffee company. MethodsX

thanks the reviewers of this article (Giovanni Caprioli and a second reviewer who would like to remain anonymous)for taking the time to provide valuable feedback.

References

[1] I. Flament, C. Chevalier, Analysis of volatile constituents of coffee aroma, Chem. Ind. (1988) 592–596.[2] T. Shibamoto, An overview of coffee aroma and flavor chemistry, in: Proceedings of the 14th International Scientific Colloquium on Coffee, San Francisco,

1991, Association Scientifique Internationale du Cafe, Paris, France, 1992, pp. 107–116.[3] Production and consumption, Trouble brewing: the changing face of coffee production, 2006 http://pubs.wri.org/pubscontent_text.cfm?ContentID=1445

(accessed 4.11.08).[4] B. Schilter, C. Cavin, A. Tritscher, A. Constable, in: Coffee, R.J. Clarke, O.G. Vizthum (Eds.), Health Effects and Safety Considerations, Blackwell Science, Oxford,

UK, 2001, pp. 165–183.[5] R.S. Sandler, Diet and cancer, Nutr. Cancer 4 (1983) 273–279.[6] G. Caprioli, S. Logrippo, M.G. Cahill, K.J. James, High-performance liquid chromatography LTQ-Orbitrap mass spectrometry method for tomatidine and non-

target metabolites quantification in organic and normal tomatoes, Int. J. Food Sci. Nutr. 65 (4) (2014) 465–469.[7] J.A. Carrillo, J. Benitez, CYP1A2 activity, gender and smoking, as variables influencing the toxicity of caffeine, Br. J. Clin. Pharmacol. 41 (1996) 605–608.[8] R.W. Derlet, J.C. Tseng, T.E. Albertson, Potentiation of cocaine and d-amphetamine toxicity with caffeine, Am. J. Emerg. Med. 10 (1992) 211–216.[9] D. Perrone, C.M. Donangelo, A. Farah, Fast simultaneous analysis of caffeine, trigonelline, nicotinic acid and sucrose in coffee by liquid chromatography–mass

spectrometry, Food Chem. 110 (2008) 1030–1035.[10] X. Goxiang, Y. Mao, Y. Yan, N. Mingming, S. Hua, M. Huang, Z. Aihua, Xiaojiao Z., C. Tianlu, J. Wei, Characterization of Pu-erh tea using chemical and metabolic

profiling approaches, J. Agric. Food Chem. 57 (2009) 3046–3054.[11] R. Hertz-Schunemann, R. Dorfner, C. Yeretzian, T. Streibel, R. Zimmermann, On-line process monitoring of coffee roasting by resonant laser ionisation time-

of-flight mass spectrometry: bridging the gap from industrial batch roasting to flavour formation inside an individual coffee bean, J. Mass Spectrom. 48(2013) 1253–1265.

[12] O. Kenny, T.J. Smyth, C.M. Hewage, N.P. Brunton1, Antioxidant properties and quantitative UPLC–MS/MS analysis of phenolic compounds in dandelion(Taraxacumofficinale) root extracts, Free Radic. Antioxid. 4 (January–June (1)) (2014) 55.